Materials used for implantation in the human body to replace damaged or diseased tissue must be biocompatible and mechanically suitable for their intended use. Metallic and polymeric materials for biomedical applications present many problems due to their high Young""s modulus (compared with that of bone), the formation of a non-adherent fibrous capsule (the resulting movement of which can lead to deterioration in function of the implant), or sometimes to their degradation products.
There is an increasing clinical use of bioactive glass and glass-ceramics because they offer the possibility of improving the long-term survivability of prostheses and improved repair of aged, diseased or damaged bone. These materials tend to form mechanically strong bonds to bone by means of a series of chemical reactions at the bone-implant interface. One of the major advantages of using a bioactive glass is the ability to control the surface chemistry, and in doing so, exerting control over the rate of bonding to the tissue.
Many biocompatible and bioactive biomaterials have been implanted. Associated problems of infections due to the intrinsic nature of an illness and to surgical intervention can arise as a consequence of implantation, even with currently aseptic surgical procedures.
Bioglass(copyright) is one example of a biocompatible material used to prepare implants. Bioglass(copyright) is often used to repair damage caused in bones, teeth, and skin where the potential for bacterial or mycotic infections is always present. An important example is osteomyelitis, one of the most dangerous diseases which is caused, in the majority of cases, by S. aureus, Salmonella or K. kingea (in children).
Even in cases of non-infectious diseases, post-operative conditions often require antibiotic treatment, which is usually administered orally. Unfortunately, this can cause bacteriological resistance to the drug, and often depletes the benign microbial flora normally present in the body, leading to gastrointestinal side effects.
Recent efforts have been focused on developing modified implant materials with antibacterial properties. Such implant materials must have suitable mechanical and chemical properties for their intended use. It would be advantageous to provide additional implant materials with antibacterial properties. The present invention provides such materials.
Silver-containing sol-gel derived bioactive glass compositions and methods of preparation and use thereof are disclosed. The compositions can be in the form of fibers, which can have any diameter between 1 xcexc and 150 xcexcm and can be either continuous or discontinuous or particles which can have any diameter for example, from 0.5 xcexcm to 3 mm, or coatings which can have thicknesses, for example, from 0.05 to 100 xcexcm. The bioactive glass (bioglass) used in the compositions includes various salts in the following ranges (weight percent of the bioglass composition):
The fibers can be woven into mats and used to make structures useful, for example as bone graft substitutes and coverings for bony defects. The fibers can also be used to make three dimensional structures for preforms to be impregnated with polymers, for example biodegradable polymers. Such structures can be linked, covalently or ionically, to bioactive compounds, for example growth factors, antibiotics, antivirals, nutrients and the like, to enhance tissue repair and promote healing.
The compositions, preferably in the form of fibers or particles, can be incorporated into implanted materials such as prosthetic implants, sutures, stents, screws, plates, tubes, and the like. The compositions in the form of particles can be applied as bioactive layers on prosthetic implants. The compositions in the form of bioactive sol-gel coatings can be applied on the surface or in the pores of prosthetic implants of various configurations.
The compositions are also useful for tissue engineering applications. An advantage of using these compositions is that anti-bacterial properties can also be imparted to devices used for in vitro and ex vivo cell culture when the compositions are incorporated into tissue engineering devices.
Silver-containing sol-gel derived bioactive glass compositions and methods of preparation and use thereof are disclosed. The compositions can be in the form of fibers, which can have any diameter between 1 xcexc and 150 xcexcm and can be either continuous or discontinuous, particles which can have any diameter, for example from 0.5 xcexcm to 3 mm, or coatings which can have thicknesses, for example from 0.05 to 100 xcexcm. The compositions are prepared using a sol-gel method and can be used for a variety of medical uses, for example bone repair, biodegradable sutures, and tissue engineering applications.
As used herein the terms xe2x80x9cbioactive glassxe2x80x9d or xe2x80x9cbiologically active glassxe2x80x9d mean an inorganic glass material having an oxide of silicon as its major component and which is capable of bonding with growing tissue when reacted with physiological fluids.
Bioactive glasses are well known to those skilled in the art, and are disclosed, for example, in An Introduction to Bioceramics, L. Hench and J. Wilson, eds. World Scientific, New Jersey (1993), the contents of which are hereby incorporated by reference.
The glass preferably includes between 40 and 90% by weight of silicon dioxide (SiO2), between about 6 and 50% by weight calcium oxide (CaO), between about 0 and 12% by weight phosphorus oxide (P2O5) and between about 0.1 and 12% by weight silver oxide (Ag2O). More preferably, the glass includes between 45 and 86% by weight of silicon dioxide oxide (SiO2), between about 10 and 36% by weight calcium oxide (CaO), between about 3 and 12% by weight phosphorus oxide (P2O5) and between about 3 and 12% by weight silver oxide (Ag2O).
CaF2, B2O3, Al2O3, MgO and K2O, Na2O may be included in the composition in addition to silicon, sodium, phosphorus and calcium oxides. Other silver salts than silver oxide can optionally be used. The preferred range for B2O3 is between 0 and 10% by weight. The preferred range for K2O is between 0 and 8% by weight. The preferred range for Na2O is between 0 and 20% by weight. The preferred range for MgO is between 0 and 5% by weight. The preferred range for Al2O3 is between 0 and 3% by weight.
It is preferred to use reagent grade glass, especially since the glass is used to prepare materials which ultimately may be administered to a patient.
In a preferred embodiment, the silver-containing, sol-gel derived bioactive glass is formed from various salts in the following ranges (weight percent of the bioglass composition):
Examples of preferred sol-gel derived bioactive glasses are shown below in Table 1, any of which can be modified to include an effective, anti-bacterial amount of silver ions using the methods described herein.
Higher CaO contents provide larger pore volumes and the onset of hydroxycarbonate apatite (HCA) deposition is accelerated. Gel-glasses with higher SiO2 contents tend to have larger surface areas and exhibit higher growth rates of formation of an HCA layer.
Silver Salts
Any suitable silver salt can be used which can be incorporated into the bioactive glasses using a sol-gel method. Silver oxide is a preferred salt. Other suitable salts include silver nitrate, silver acetate, silver bromide and silver chloride. The amount of silver in the compositions is generally in the range of between about 0.1 and 12 percent by weight, preferably between about 3 and 12 percent by weight.
The toxicity limit for the ingestion of soluble silver salts is about 1 gram for humans, but it is not generally considered a threat to life, as an accidental exposure to high doses of silver is extremely rare. Indiscriminate use of silver-containing pharmaceutical preparations and devices can lead to toxic reactions such as argyria. The term xe2x80x9ceffective, antibacterial amountxe2x80x9d of silver refers to an amount effective to significantly reduce the amount of bacteria in an area proximate to where the bioactive glass is present. This amount would be expected to vary depending on a variety of factors, including the type of bacteria, the bacterial concentration, the type of media and the intended use. Those of skill in the art can readily determine an appropriate, antibacterial amount of silver to use. The bioactive glass compositions can be adjusted to include a variety of concentrations of silver ions.
The antimicrobial action of silver has been verified on a number of gram positive and gram negative bacteria, and fungi, among which are E. coli, P. aeruginosa, S. epidermis, C albicans. The enzymes on which the inactivating influence has been studied include; urocinase, xcex2-galactosidase, phosphomannose-isomerase, and several oxygenases. It has been postulated that silver exerts its toxicity at multiple sites, among which are the respiratory chain, the phosphate uptake and storage, and the cell wall synthesis. The overall result of these alterations is a lethal leakage of metabolites from the cell, including phosphate and potassium (K+).
The mechanism of action of Ag+is believed to be related to its complexation to membranes, enzymes and other cellular components. The silver ion is strongly chelated by electron donor groups such as amines, hydroxyls, phosphate and thiols. The latter are thought to be the most important chelating groups, according to microbiological, biochemical and electrochemical data. The silver ion is believed to interact with protein molecules via exposed cysteine residues.
Sol-Gel Method
The compositions are prepared using a sol-gel method. When compared with conventional glass production techniques, there are a number of advantages associated with the sol-gel process: lower processing temperatures, purer and more homogenous materials, good control over the final composition, and tailoring of the surface and pore characteristics of the product.
Alkoxide derived gel-glasses of the system SiO2xe2x80x94CaOxe2x80x94P2O5 present an expanded compositional range of bioactivity over bioactive glasses made by melt processes. The difference in bioactive behavior relates directly to the structure of sol-gel derived materials. The gel-glasses have a much higher surface area, a higher concentration of silanol groups per unit area on the surface and a higher concentration of metastable three and four membered siloxane rings. The bioactivity is influenced by the texture of the material as well as the chemical composition.
The gel-glasses are ideally suited as bone graft materials, due to their higher resorption rates in vitro and in vivo. Furthermore, the rate of soluble silicon species released during HCA formation, and the stimulation of bone growth are improved compared with those of the melt-derived bioactive glass.
The compositions can be prepared, for example, by synthesizing an inorganic network by mixing metal alkoxides in solution, followed by hydrolysis, gelation, and firing to produce a porous matrix or a dense glass. The firing can be done at relatively high temperatures (600-1100xc2x0 C.), and can also be done at low temperatures (on the order of about 200 to 250xc2x0 C.). The sol-gel process used herein uses a four or more component system, including at least SiO2, CaO, P2O5 and a silver salt, for example, Ag2O. In one embodiment, the silver-containing sol-gel bioactive glass is produced as a gel network from tetraethoxysilane (TEOS), phosphorous alkoxide, calcium nitrate and silver oxide in water-ethanol solution.
The process, and the types of reactions which typically occur in sol-gel formation, is described in more detail below.
Aqueous Solutions of SiO2 
The first step of the sol-gel process typically involves mixing the precursor, silicon alkoxide, solvent (generally water), and an acid or alkaline catalyst. This step can dramatically affect the homogeneity of a multi-component gel, which is also influenced by the nature and reactivity of the precursors, the nature and solubility of the reactants in the selected solvent, the concentration of the selected solvent, the sequence of addition, the pH and the time and temperature of the reaction.
After mixing, the alkoxide precursor is hydrolyzed to silicic acid, which then condenses to yield the silica gel. The hydrolysis reactions are shown below, where R is an alkyl group:
Si(OR)4+4H2O⇄Si(OH)4+4ROH
ROxe2x80x94Si(OR)3+HOxe2x80x94Si(OR)3⇄(OR)3Sixe2x80x94Oxe2x80x94Si(OR)3+ROH
HOxe2x80x94Si(OR)3+HOxe2x80x94Si(OR)3⇄(OR)3Sixe2x80x94Oxe2x80x94Si(OR)3+H2O
Hydrolysis is believed to occur via bimolecular nucleophilic attack (SN2) of water on the Si atom, and can be catalyzed by acids or bases. The nature of the alkoxide group (R) influences the rate of hydrolysis through inductive and steric effects. When R is an electron-withdrawing group it accelerates the reaction, and if the R group is bulky it slows down the reaction rate.
When producing a multi-component system, TEOS, rather than tetramethoxysilane (TMOS) (which only takes minutes to hydrolyze), is the chosen precursor in order to have better control on the rate of hydrolysis. Tetraethoxyphosphate (TEP) is used as a convenient source of phosphate monomers. Soluble metal salts, like nitrates, can also be used to introduce modifier atoms. The condensation is catalyzed by the same catalysts used in the hydrolysis and its reaction rate changes with the pH value of the solution.
The pH conditions also affect how coarse and consolidated the resulting gel will be. The shape and the size of polymeric structural units are determined by the relative values of the rate constants for hydrolysis and polycondensation reactions. Fast hydrolysis and slow condensation favor formation of linear polymers; on the other hand, slow hydrolysis and fast polymerization results in larger, bulkier polymers.
As the sol particles grow and collide condensation occurs and macroparticles form. The sol becomes a gel when it can support a stress elastically.
Aging
The aging step involves maintaining the cast object for a period of time (typically from hours to days) immersed in liquid. During aging, polycondensation continues along with localized dissolution and re-precipitation, until free reactive species and reactive sites have all reacted. This process, that decreases the porosity and strengthens the gel, is called syneresis; it causes the gel to shrink and expel the pore liquors.
Along with syneresis, another phenomenon, called Ostwald ripening, takes place. This is an irreversible process involving preferential dissolution of high potential energy convex surfaces, followed by deposition on low energy concave surfaces. Thus, necks begin to form between primary particles and smaller pores are filled at the expense of the bigger ones. This coarsening process is usually slower compared with the syneresis, but it can affect the texture of the gel, particularly when the aging takes place at high temperatures or high pH values.
Drying
During drying, the liquid is removed from the interconnected pore network. Strong capillary stresses can develop and cause the gel to crack unless the drying process is controlled by decreasing the solid-liquid interfacial tension. This can be achieved in a number of ways: by addition of surfactants; by hypercritical evaporation which eliminates the solid-liquid interface; or by obtaining monodisperse pore sizes from controlling the rates of hydrolysis and condensation.
Stabilization and Densification
Removal of the surface silanol groups results in a chemically stable porous material. This can be achieved using thermal and/or chemical methods. Chemical methods often involve modification of the silica surface by replacing the silanol groups with more hydrophobic and less reactive species (e.g., chlorides and fluorides). Heating above 400xc2x0 C. results in an irreversible dehydration due to the increasing elimination of isolated silanol groups and to the structural relaxation which takes place.
In multi-component systems the calcination process also serves to degrade other species that are present in the gel (e.g., calcium nitrate, Ca(NO3)2). Nitrate species are undesirable in bioapplications, and are also a source of in-homogeneity. They also remain in the specimen after drying and must be decomposed. Note that the decomposition of pure Ca(NO3)2 occurs at 561xc2x0 C. Hence this temperature must be exceeded during successful stabilization if such groups are present.
Heating at temperatures between 800xc2x0 C. and 1500xc2x0 C. (depending on the initial porosity, interconnectivity, atmosphere, and composition) will densify the gel to become a consolidated glass with a density substantially equivalent to that of glasses made by conventional melting and casting.
Adjusting the Pore Volume of the Compositions
Suitable pore diameters are between 20 and 400 xc3x85. Pore diameters larger than 0.1 microns can be achieved using a sintering and/or foaming processes. The sintered structure may then be impregnated with a variety of materials, as discussed in more detail below.
To aid in preparing glass compositions with high porosity, the glass composition can include a material which can be preferably leached out of the glass composition, and, in doing so, provide the composition with high porosity. For example, minute particles of a material capable of being dissolved in a suitable solvent, acid, or base can be mixed with or incorporated into the glass, and subsequently leached out. The resulting voids have roughly the same size as the particle that was leached out. The size of the pores and degree of porosity also depends on the amount of added material relative to the amount of glass. For example, if the leached material constituted about 80% of the glass, then the glass would be approximately 80% porous when the material was leached out. When leaching the glass composition, care should be taken not to leach out a significant amount of those components which add to the bioactivity of the glass, i.e., the calcium and phosphorus oxides, or the antibacterial properties of the glass, i.e., the silver ions.
Preparation of Bioactive Glass Fibers
Continuous fibers can be prepared, for example, by extruding the sol through a spinneret. The fibers can then be aged, dried, and thermally stabilized. Long fibers may be woven into a mesh, short fibers may be combined by mixing them with a degradable adhesive, such as a solution of carboxymethylcellulose (CMC). The resulting material is then heated in a kiln to sinter the material and burn off the binder.
Preparation of Bioactive Glass Coatings
Coatings can be prepared using means well known to those of skill in the art, including dipping an article to be coated into an appropriate sol-gel solution which is then treated to form the sol gel, and spraying the article to be coated with particles of the bioactive glass.
B. Methods of Shaping the Fiber into Desired Structures
After the composition has been spun into a fiber, for example in a spinneret, the resulting fiber can be shaped into desired structures. In one embodiment, the fiber is merely wound and can be used as a degradable suture material. In other embodiments, the fiber is mixed with various additional components, including polymeric materials, and shaped into desired articles of manufacture. The shaping can be performed via any acceptable means, including laser ablation, extrusion, molding techniques, and the like.
The fibers can be formed into a mesh or fabric (woven or non-woven). The mesh can be used, for example, in wound healing and wound covering. In one embodiment, the fibers are woven into mats or other structures. The resulting material can be used to make structures useful, for example as bone graft substitutes and coverings for bony defects.
The fibers can also be used to make three dimensional structures for preforms to be impregnated with polymers, for example biodegradable polymers. Such structures can be linked, covalently or ionically, to bioactive compounds, for example, growth factors, antibiotics, antivirals, nutrients and the like to enhance tissue repair and promote healing.
The fibers can be incorporated into implanted materials, such as prosthetic implants, sutures, stents, screws, plates, tubes, and the like.
The fibers (as well as particles) are also useful for tissue engineering applications. An advantage of using these fibers is that anti-bacterial properties can also be imparted to devices used for in vitro and ex vivo cell culture when the fibers are incorporated into tissue engineering devices.
When the fiber has a relatively high porosity, it has a relatively fast degradation rate and high surface area, in comparison to non-porous bioactive glass fiber compositions. The degree of porosity of the glass is between about 0 and 85%, preferably between about 10 and 80%, and more preferably, between about 30 and 60%.
The form of the bioactive glass (particles, fibers, and the like) depends in large part on the intended use of the compositions. Those of skill in the art can readily select an appropriate form for the bioactive glass for an intended use. Examples of applications of the silver-containing sol-gel derived bioactive glass compositions described herein include surgical treatment of periodontal and osteoinfections, inclusion in preparations to cure skin infections, use as a preservative in cosmetic preparations, introduction as antimicrobial agent in health care products and in detergents, and as preventative antimicrobial agents for surgery that involves the use of implanted biomaterials and/or devices.
In addition to the bioactive glass composition, the formulations can include other therapeutic agents such as antibiotics, antivirals, healing promotion agents, anti-inflammatory agents, immunosuppressants, growth factors, anti-metabolites, cell adhesion molecules (CAMs), bone morphogenic proteins (BMPs), vascularizing agents, anti-coagulants, and topical anesthetics/analgesics.
The antibiotics can be topical antibiotics suitable for skin treatment. Examples of such antibiotics include but are not limited to: chloramphenicol, chlortetracycline, clyndamycin, clioquinol, erythromycin, framycetin, gramicidin, fusidic acid, gentamicin, mafenide, mupiroicin, neomycin, polymyxin B, bacitracin, silver sulfadiazine, tetracycline and chlortetracycline.
Suitable antivirals include topical antivirals, such as acyclovir, and gancyclovir. Suitable anti-inflammatory agents include corticosteroids, hydrocortisone and nonsteroidal antinflammatory drugs. Suitable growth factors include basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), transforming growth factors xcex1 and xcex2 (TGF xcex1 and xcex2), platelet-derived growth factor (PDGF), and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF)). Suitable topical anesthetics include benzocaine and lidocaine.
In one embodiment, the therapeutic agent is one which would otherwise cause an inflammation at the site at which it is delivered, and the bioactive glass compositions reduce the associated inflammation. For example, a number of compounds, for example, amine compounds, result in inflammation when administered topically, i.e., in a transdermal patch.
In addition, the bioactive glass may be combined with any biocompatible material, such as biodegradable polymer like polylactic/glycolic acid to form a composite material for accelerating wound healing.
The proportion of other therapeutic agents varies according to the agent and the nature of the application. However, the preferred proportions are such that the amount of the agent administered to the patient is in a dosage range accepted within standard medical care.
The silver-containing, sol-gel derived bioactive glass compositions can be incorporated into implanted materials, such as prosthetic implants, sheets, pins, valves, sutures, stents, screws, plates, tubes, and the like, by incorporating bioactive glass particles into the implanted materials. The compositions can be moldable or machinable.
Table 2, below, shows a relation between the form of the bioactive glass composition and the intended function. This table is not intended to limit the type of form which can be used for an intended function, merely to exemplify types of forms and matching functions. The compositions described herein can be in any of these forms.
The articles of manufacture are imparted with anti-bacterial properties via the incorporation of the silver ions into the bioactive glass, which will allow the articles to be implanted, or used to culture cells, with a reduced likelihood of bacteriological contamination.
Cell Growth and Culture
There are many solutions used for culturing cells. These include Dulbecco""s minimal essential media, Hank""s balanced salt solution, and others. These solutions are essentially isotonic with the cells to be cultured. A problem associated with cell culture is often the growth of bacteria in culture along with the desired cells. Bacterial growth can be minimized by incorporating the bioactive glass compositions into matrices used in cell culture and tissue engineering applications.
The silver-containing, sol-gel derived bioactive glass fibers, particles and/or coatings are capable of dramatically reducing the amount of time necessary for wound healing to occur. Implants including the fibers, particles or coatings, preferably highly porous fibers or particles, alone or in combination with other anti-bacterial agents, can augment the natural healing process. The effectiveness of the fibers, particles and coatings described herein is most dramatically illustrated in immune compromised patients whose ability to heal wounds is somewhat suppressed.
In one embodiment, the fibers and/or particles are used to fill voids, including voids created during medical procedures. For example, during a root canal operation, the hollowed-out tooth can be filled with a composition including bioactive glass fibers and/or particles. This will help prevent bacterial infection until the tooth is ultimately filled. Also, bioglass-containing compositions can be used to fill the pockets that can develop between the teeth and gums. Compositions including bioactive glass fibers and/or particles can be used to fill voids present in aneurysms, and prevent bacterial growth inside the filled void. Other voids which can be filled include those formed surgically, such as removal of a spleen, ovary, gall bladder, or tumor.
The methods for grafting skin involve applying meshes or fabrics including the silver-containing, sol-gel derived bioactive glass fibers, particles and/or coatings to either the graft site or donor tissue before it is placed in its intended location. Those interested in a detailed description of skin grafting are referred to xe2x80x9cSkin Grafts,xe2x80x9d in Selected Readings in Plastic Surgery, Vol. 7, no. 2, P. L. Kelton, MD, Baylor University Medical Center (1992). The graft may also be further treated with a topical carrier prior to placement. The application of bioactive glass to grafts is intended to increase the likelihood that the graft will xe2x80x9ctakexe2x80x9d and incorporate in the host bed. It is intended that the bioactive glass will act as an intermediary bond between the host and graft tissue, suppress the overall inflammatory response which could lead to rejection, as well as accelerate the overall healing process which will lead to a faster and more successful acceptance.
The bioactive glass fibers, particles and/or coatings can be administered locally to a surgical site to minimize post-surgical adhesions. The compositions can optionally be incorporated into a polymeric material which is applied to the surgical site. Alternatively, the bioactive gel-glass can be used as a coating on polymeric materials which is applied to the surgical site. Preferably, the polymeric material is biodegradable. Suitable polymeric materials for this purpose are disclosed, for example, in U.S. Pat. No. 5,410,016 to Hubbell et al., the contents of which are hereby incorporated by reference. Other materials suitable for this purpose, such as Interceed(copyright), agarose and crosslinked alginate, are well known to those of skill in the art.
Biomedical implants are often associated with inflammation at the site of implantation. Incorporation of the bioactive glass fibers described herein, in particular, the highly porous bioactive glass fibers, into the implants, especially on the surface of the implants, can greatly reduce the inflammation associated with the implants. This can be especially useful in suture materials to minimize the inflammation associated with these materials. The anti-bacterial properties of the compositions also allow the sutures to minimize the infection surrounding the suture site.